Venereal syphilis is caused by T. pallidum subsp. pallidum, and other human treponemal diseases are caused by the closely related organisms T. pallidum subsp. pertenue (yaws), T. pallidum subsp. endemicum (bejel or endemic syphilis), and T. carateum (pinta). Another close relative, T. paraluiscuniculi, gives rise to venereal spirochetosis in rabbits. Venereal syphilis is still a major health problem in the United States and other countries, and the other treponematoses have reemerged in tropical areas of the world. Although these organisms are largely indistinguishable by morphology, DNA and protein content, and antigenicity of well-characterized proteins, there are definite differences in terms of the clinical manifestations and cross-immunity obtained with these organisms. In this new application, Dr. Lukehart and coworkers propose to take advantage of these differences to identify subspecies pallidum-specific genes and gene products that are likely to contribute to specific immunity to syphilitic infection and to the unique pattern of syphilis pathogenesis. In Specific Aim 1, a subsp. pallidum (Nichols strain) expression library will be screened with specific antisera against different pathogenic treponemal species and subspecies, as well as with antisera adsorbed extensively with other pathogenic treponemes to remove cross-reactive antibodies. In addition, a subtractive hybridization technique utilizing adapter-mediated amplification will be employed to isolate sequences present in subsp. pallidum but absent from subsp. pertenue or T. paraluiscuniculi. Clones identified by these methods will then be screened for subsp. pallidum specificity by hybridization, and selected clones will be sequenced for homology searches (Specific Aim 2). Clones will be assigned priorities according to probable outer membrane location and homology with known virulence factors, and full-length genes for each ORF will be obtained from a genomic library or by PCR amplification of the ORF using primer sequences from the T. pallidum genome sequence when it becomes available. In Specific Aim 3, sequence-specific probes and monospecific antisera will be utilized to determine the presence and expression of the subspecies-specific genes in a bank of 26 subsp. pallidum, 4 subsp. pertenue, 2 subsp. endemicum, and 4 T. paraluiscuniculi isolates. The immune response to the subspecies-specific proteins during the course of syphilitic infection in humans and experimentally infected rabbits will then be determined and related to the gradual development of immunity that is known to occur during infection (Specific Aim 4). Finally, in Specific Aim 5, the ability of the subsp. pallidum-specific proteins to provide protective immunity will be evaluated in a rabbit model. In preliminary studies, Dr. Lukehart and colleagues have a) selected and demonstrated varying degrees of cross-immunity among three strains of subspecies pallidum, subspecies pertenue, and T. paraluiscuniculi; b) described a bank of 37 pathogenic treponeme strains maintained in their laboratory; c) provided preliminary data on the construction of expression and subtractive hybridization libraries, and the differential screening of libraries using opsonic antisera; d) used 16S-23S intergenic regions, 15 kd and 17 kd antigen genes, and 15 kd antigen gene untranslated regions in an attempt to identify suspecies- and species-specific genetic differences; e) expression and purification of recombinant T. pallidum antigens; and f) production of hyperimmune serum and use of recombinant proteins for immunization and evaluation of immune responses. Supplemental information provided after submission indicates that two subtractive hybridization clones a) have homology to the Treponema denticola major outer surface protein (Msp); b) hybridize with multiple restriction fragments in T. pallidum subsp. pallidum DNA, suggestive of the presence of at least 3 copies of related sequences; c) exhibit sequence heterogeneity among treponemal species and subspecies, as determined by the lack of amplification from subsp. endemicum and T. paraluiscuniculi genomic DNA and comparison of subsp. pallidum and subsp. pertenue sequences.